The possible deployment of organophosphate (OP) nerve agents in terrorist actions is of immediate concern and has prompted new investigations to develop therapeutics to combat human exposures. These research endeavors are producing new approaches and small molecule discoveries to counteract OP poisoning that are nearing initial translational applications. Our long-term objective is to develop translational assays that evaluate, measure and validate new OP therapeutic agents in live subjects over time by employing positron emission tomography (PET) imaging. PET imaging allows the assessment of key pharmacokinetic (PK) and pharmacodynamic (PD) parameters in the presence and absence of therapeutic treatments and antidote agents. Thus, the goal of this application is to establish new OP PET imaging tracers, to demonstrate their initial functional imaging utility in live rodent subjects, and to initially validate their performance qualities in the presence of specific countermeasure treatments. To satisfy this goal we will establish requisite OP PET imaging tracers using a logical discovery paradigm that will advance rationally designed alkoxy methylphosphonate (AMP) and ethyl 2-methoxyethyl phosphonates (EMP) radioligands with the following five progressive and collaborative specific aims: Aim 1: Prepare 10 non-radioactive (cold) high purity specific AEP derivatives, demonstrate they act similarly to OP nerve agents by evaluating the anti-cholinesterase activity and mechanism, and rank their inhibitory anti-cholinesterase activity profiles. Aim 2: Select the top five Aim 1 candidate agents (inhibitor ki values e 104 M-1min-1) and prepare their positron labeling carbon-11 and fluorine- 18 precursors. Aim 3: Produce five carbon-11 and fluorine-18 radiolabeled AMP inhibitor tracers (radiochemical yields e 10 %, high specific activity). Aim 4: Evaluate CNS tissue PK qualities of the Aim 3 tracers with microPET-CT-MR imaging in rodent subjects, rank the tracers as a function of specific tissue tracer penetration and cognate time-activity curve (TAC) qualities (uptake, maximum and washout) then select the 3 best tracers (penetration e 1 % injected dose and TACs shown with maximums and initial washout phases within 4-5 half-lives of the positron radiolabel) to advance to Aim 5. Aim 5: Establish translational proof-of-concept by discerning which of the three Aim 4 tracers are suitable for advancement by performing discrete tracer PD microPET imaging experiments carried out with the co-administration of select known OP agents, including the established: a) OP inhibitor paraoxon, and b) OP poisoning antidote treatment, 2-pyridine aldoxime methiodide (2-PAM). PUBLIC HEALTH RELEVANCE: The United States remain vulnerable to acts of terrorism using unconventional weapons including highly toxic chemical nerve agents like organophosphates, which are relatively inexpensive to produce and deploy in an attack on civilians. The relevance of this investigation is to develop novel positron emission tomography (PET) tissue imaging agents based on organophosphate structure to be used to demonstrate the in vivo action and distribution of organophosphates, and serve to validating and assess new therapeutic drugs and approaches for clinical use in the event of an attack.